steroids 73 (2008) 1160–1173 available at www.sciencedirect.com journal homepage: www.elsevier.com/locate/steroids Heterologous expression of human mPR␣, mPR␤ and mPR␥ in yeast confirms their ability to function as membrane progesterone receptors Jessica L. Smith, Brian R. Kupchak, Ibon Garitaonandia 1, L. Kim Hoang, Andrew S. Maina, Lisa M. Regalla 2, Thomas J. Lyons ∗ Department of Chemistry, University of Florida, P.O. Box 117200, Gainesville, FL 32611, United States article info abstract Article history: The nuclear progesterone receptor (nPR) mediates many of the physiological effects of Received 1 April 2008 progesterone by regulating the expression of genes, however, progesterone also exerts Received in revised form 6 May 2008 non-transcriptional (non-genomic) effects that have been proposed to rely on a receptor Accepted 8 May 2008 that is distinct from nPR. Several members of the progestin and AdipoQ-Receptor (PAQR) Published on line 18 May 2008 family were recently identified as potential mediators of these non-genomic effects. Mem- branes from cells expressing these proteins, called mPR␣, mPR␤ and mPR␥, were shown to Keywords: specifically bind progesterone and have G-protein coupled receptor (GPCR) characteristics, Membrane progestin receptors although other studies dispute these findings. To clarify the role of these mPRs in non- Progesterone genomic progesterone signaling, we established an assay for PAQR functional evaluation Yeast using heterologous expression in Saccharomyces cerevisiae. Using this assay, we demonstrate ␣ ␤ ␥ Adiponectin unequivocally that mPR , mPR and mPR can sense and respond to progesterone with EC50 Osmotin values that are physiologically relevant. Agonist profiles also show that mPR␣, mPR␤ and mPR␥ are activated by ligands, such as 17␣-hydroxyprogesterone, that are known to activate non-genomic pathways but not nPR. These results strongly suggest that these receptors may indeed function as the long-sought-after membrane progesterone receptors. Additionally, we show that two uncharacterized PAQRs, PAQR6 and PAQR9, are also capable of respond- ing to progesterone. These mPR-like PAQRs have been renamed as mPR␦ (PAQR6) and mPR␧ (PAQR9). Additional characterization of mPR␥ and mPR␣ indicates that their progesterone- dependent signaling in yeast does not require heterotrimeric G-proteins, thus calling into question the characterization of the mPRs as a novel class of G-protein coupled receptor. © 2008 Elsevier Inc. All rights reserved. 1. Introduction This type of signal transduction is often referred to as genomic signaling because this receptor, also called the nuclear proges- The classic paradigm for progesterone-mediated signal trans- terone receptor (nPR), doubles as a DNA binding transcription duction involves diffusion of the steroid hormone into cells factor and, as such, modulates gene transcription in response and binding to soluble intracellular progesterone receptors [1]. to progesterone. It has long been recognized, however, that ∗ Corresponding author. Tel.: +1 352 846 3392; fax: +1 352 846 2095. E-mail address: [email protected]fl.edu (T.J. Lyons). 1 Current address: Department of Neurobiology, SBR-14, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, United States. 2 Current address: Boston Museum of Science, Science Park, Boston, MA 02114, United States. 0039-128X/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.steroids.2008.05.003 steroids 73 (2008) 1160–1173 1161 many of the physiological effects of progesterone occur far too attribute progesterone-dependent effects solely to one pro- rapidly to require gene transcription and often occur in cells tein. This line of research is beyond the scope of this study that are transcriptionally silent, such as sperm [2]. Moreover, and, ultimately, the discovery of the true physiological roles the receptor(s) responsible for mediating the non-genomic of mPR␣,mPR␤ and mPR␥ may require the development of effects of progesterone have a markedly different agonist pro- mouse knockout strains. files from nPR [3], suggesting that it is a distinct receptor. Herein, we address another important aspect of character- These facts have led researchers to propose the existence of izing mPR␣,mPR␤ and mPR␥—their biochemistry. To date, the an integral membrane progesterone receptor (mPR) that binds biochemical characterization of mPR␣,mPR␤ and mPR␥ has progesterone at the cell surface and rapidly generates intracel- involved their expression in either vertebrate cell culture or lular second messengers [4]. This mechanism for progesterone E. coli [19,10,11]. Expression in vertebrate cell culture has been response has been called non-genomic signaling because it fruitful, however, conflicting results were obtained by differ- does not absolutely require transcription to proceed, although ent groups [17,19]. Moreover, this approach is limited by the there is no reason to suspect that non-genomic mechanisms aforementioned overabundance of progesterone-binding pro- do not also regulate transcription [5]. The existence of mPR is teins in vertebrate cells that make data interpretation difficult. a matter of intense debate. Indeed, it has been argued that On the other hand, E. coli do not contain known progesterone there is no need to invoke the existence of a distinct mPR receptors and heterologous expression in this organism has because the nPR itself is capable of mediating rapid non- been successfully used to confirm progesterone binding to genomic effects by binding to and regulating the activity of isolated mPRs embedded in intact E. coli membranes [10,11]. other signaling proteins [6–9]. Nevertheless, several candidate However, while expression in E. coli is certainly useful for mPRs have emerged and, while it is clear that some of these studying progesterone binding to these receptors, it cannot candidates bind progesterone, it is not clear how, or even if, yet be used to investigate functionality, since E. coli it is not they function as receptors to mediate the non-genomic effects known if PAQR receptors function the same in this organism of progesterone. as they do in eukaryotes. Recently, a series of seminal papers were published iden- Thus, a resolution to the debate about whether mPR␣, tifying three integral membrane proteins from fish that not mPR␤ and mPR␥ can sense and respond to progesterone only bind progesterone, but also seem to mediate many of requires a system with two fundamental properties. First, its rapid non-genomic effects [10,11]. These three proteins, such a system must be devoid of known progesterone- renamed mPR␣,mPR␤ and mPR␥, are conserved in vertebrates binding/sensing proteins so that the mPRs can be studied in and belong to a newly characterized family of proteins that isolation. Second, this system must have an intact signaling includes the yeast osmotin receptor (Izh2p) [12] and human apparatus that allows for monitoring signal transduction in adiponectin receptors (AdipoR) [13]. This family is known as response to progesterone. Herein we report the development the progestin- and AdipoQ-Receptor (PAQR) family [14]. of such a system that entails the heterologous expression of A series of follow-up studies produced compelling evidence mPR␣,mPR␤ and mPR␥ in the yeast Saccharomyces cerevisiae. that mPR␣,mPR␤ and mPR␥ function as membrane proges- This choice of model systems is advantageous for several rea- terone receptors. However, as a recent review in this journal sons. First, yeast is a eukaryotic system for which simple yet demonstrates [15], the field remains embroiled in controversy. powerful genetic tools exist. Second, yeast possesses receptors The primary reason for this is the fact that a recent study was in the PAQR family suggesting that the machinery required to unable to reproduce the original results [16,17]. Another signif- read second messengers produced by these proteins is present icant source of contention is the assertion that these receptors [27]. Third, S. cerevisiae neither makes nor uses progesterone. function as a new class of G-protein-coupled receptor (GPCR) In fact, a recent publication showed that massive doses of [18,19,10,11] despite the fact that no other class of PAQR seems progesterone (1 mM) did little more than weakly induce the to couple to G-proteins and that members of the PAQR family general stress–response [28]. The low background biological of receptors bear only superficial similarity to GPCRs. activity of progesterone in this system makes it an ideal model Further investigation is needed before mPR␣,mPR␤ and for the study of individual progesterone receptors in a living mPR␥ can be universally accepted as membrane progesterone cell. Indeed, S. cerevisiae has already been successfully adapted receptors. One side of this equation involves the study of the for the biochemical characterization of nuclear progesterone physiological roles of these proteins in vivo. To date, no stud- receptors [29]. ies have demonstrated an unequivocal role for these proteins We recently published a study showing that the yeast in the physiology of progesterone. Part of the reason for this osmotin receptor (Izh2p) controls a signaling pathway in S. is the overabundance of progesterone-binding proteins and cerevisiae that negatively regulates the expression of a gene putative progesterone receptors in vertebrates that can poten- called FET3 [30]. The physiological importance of this reg- tially confound data analysis. Not only do vertebrate cells ulation